U.S. patent application number 10/534392 was filed with the patent office on 2006-10-19 for process for reducing the acrylamide content of heat-treated foods.
This patent application is currently assigned to BAYER CROPSCIENCE GMBH. Invention is credited to Bernd Essigmann, Claus Frohberg, Martin Quanz, Stephan Soyka.
Application Number | 20060233930 10/534392 |
Document ID | / |
Family ID | 32314723 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060233930 |
Kind Code |
A1 |
Soyka; Stephan ; et
al. |
October 19, 2006 |
Process for reducing the acrylamide content of heat-treated
foods
Abstract
The present invention relates to a process for reducing the
acrylamide content of heat-treated foods compared with
corresponding conventional heat-treated foods.
Inventors: |
Soyka; Stephan;
(Sint-Denijs-Western, BE) ; Frohberg; Claus;
(Kleinmachnow, DE) ; Quanz; Martin; (Berlin,
DE) ; Essigmann; Bernd; (Berlin, DE) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
BAYER CROPSCIENCE GMBH
BRUNINGSTRASSE 50
FRANKFURT
DE
65929
|
Family ID: |
32314723 |
Appl. No.: |
10/534392 |
Filed: |
November 7, 2003 |
PCT Filed: |
November 7, 2003 |
PCT NO: |
PCT/EP03/12476 |
371 Date: |
January 13, 2006 |
Current U.S.
Class: |
426/549 |
Current CPC
Class: |
A21D 2/36 20130101; A23V
2002/00 20130101; A23L 19/18 20160801; A23V 2002/00 20130101; A23L
19/19 20160801; A23V 2300/21 20130101; A23L 7/13 20160801 |
Class at
Publication: |
426/549 |
International
Class: |
A21D 10/00 20060101
A21D010/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2002 |
EP |
02025008.0 |
Feb 21, 2003 |
EP |
03003235.3 |
May 21, 2003 |
EP |
03090151.6 |
Claims
1. Process for reducing the acrylamide content of heat-treated
foods compared with corresponding conventional heat-treated foods
comprising: (a) selecting plant material which, compared with
corresponding conventional plant material, has a reduced content of
soluble sugars; (b) processing said plant material to give a food;
and (c) heat treating the food produced in process step b).
2. Process according to claim 1, in which the said acrylamide
content is reduced by at least 15% compared with the acrylamide
content of corresponding conventional heat-treated foods.
3. Process according to claim 1, in which said acrylamide content
is reduced by at least 30% compared with the acrylamide content of
corresponding conventional heat-treated foods.
4. Process according to claim 1, in which said heat treatment is
carried out at temperatures of at least 100.degree. C.
5. Process according to claim 1, in which said heat-treated foods
include potato chips, French fries, parfried potato chips, mashed
potato, biscuits, crackers, crisp bread, breakfast cereals, maize
crisps (tacos), popcorn, bread crisps, wafers, salt sticks, coffee,
bread, rolls, cakes, rice crisps, pizza and toast, tortillas,
croquettes, wedges, potato sticks, twisters, bread coatings for
meat, fish and vegetables, bread coatings for nuts, tortilla chips,
bread or cereal formulations, or pre-cooked meals.
6. Process according to claim 1, in which the plant material used
is genetically modified, the genetic modification leading to a
reduction in the content of soluble sugars, compared with
corresponding conventional plant material from wild type
plants.
7. Process according to claim 6, in which the genetic modification
leads to a reduction in the activity of one or more endogenous R1
proteins occurring in the plant cell compared with corresponding
plant cells of wild type plants which have not been genetically
modified.
8. Process according to claim 6, in which said genetic modification
is the introduction of one or more foreign nucleic acid molecules,
the presence and/or expression of which leads to the reduction in
the activity of one or more endogenous R1 proteins occurring in the
plant cell compared with corresponding plant cells of wild type
plants which have not been genetically modified.
9. Process according to claim 8, in which said foreign nucleic acid
molecules include (a) DNA molecules which code for at least one
antisense RNA causing a reduction in expression of endogenous genes
which code for R1 proteins; (b) DNA molecules which, via a
cosuppression effect, lead to reduction of the expression of
endogenous genes coding for R1 proteins; (c) DNA molecules which
code for at least one ribozyme which cleaves in a specific manner
transcripts of endogenous genes coding for R1 proteins; (d) nucleic
acid molecules which are introduced by means of in vivo mutagenesis
and lead to a mutation or insertion of a heterologous sequence in
genes coding for endogenous R1 proteins, the mutation or insertion
causing a reduction in the expression of said genes or the
synthesis of inactive R1 proteins; (e) DNA molecules which
simultaneously code for at least one antisense RNA and at least one
sense RNA, said antisense RNA and said sense RNA forming a
double-stranded RNA molecule which causes a reduction in the
expression of endogenous genes coding for R1 proteins; (f) DNA
molecules which contain transposons, the integration of the
transposon sequences leading to a mutation or an insertion in
endogenous genes coding for R1 proteins which causes a reduction in
the expression of said genes or the synthesis of inactive R1
proteins; or (g) T-DNA molecules which, via insertion in endogenous
genes coding for R1 protein cause a reduction in the expression of
genes coding for R1 protein or the synthesis of inactive R1
proteins.
10. Process according to claim 1, in which said plant material
originates from potato plants.
11. Process according to claim 10, in which said heat-treated foods
are potato chips, potato crisps, parfried potato chips or mashed
potato.
12. A method for producing heat-treated foods which, compared with
corresponding conventional heat-treated foods, have a reduced
acrylamide content comprising reducing the acrylamide content of
heat-treated foods according to claim 1.
13. The method according to claim 12, in which said acrylamide
content is reduced by at least 15% compared with the acrylamide
content of corresponding conventional heat-treated foods.
14. The method according to claim 12, in which said heat-treated
foods include potato chips, French fries, parfried potato chips,
mashed potato, biscuits, crackers, crisp bread, breakfast cereals,
maize crisps (tacos), popcorn, bread crisps, wafers, alts sticks,
coffee, bread, rolls, cakes, rice crisps, pizza and toast,
tortillas, croquettes, wedges, potato sticks, twisters, bread
coatings for meat, fish and vegetables, bread coatings for nuts,
tortilla chips, bread or cereal formulations, or pre-cooked
meals.
15. (canceled)
16. Process for identifying material which is suitable for
producing heat-treated foods having a reduced acrylamide content,
comprising: a) determining the content of soluble sugars and/or
amino acids of the plant material which is suitable for producing
heat-treated foods; and b) selecting such plant material according
to process step a) which, compared with corresponding conventional
plant material, has a reduced content of soluble sugars and/or
amino acids.
17. Process according to claim 1, wherein said pre-cooked meals are
baby food.
18. The method according to claim 14, wherein said pre-cooked meals
are baby food.
Description
[0001] The present invention relates to a process for reducing the
acrylamide content of heat-treated foods, compared with
conventional heat-treated foods.
[0002] Recently, the Swedish National Food Administration (NFA) and
scientists from Stockholm University have published new research
results according to which, in various foods which are given a high
heat treatment on preparation, acrylamide, a toxic and possibly
carcinogenic substance, is formed. The NFA informed other national
and international authorities and organizations in order to
stimulate international collaboration and research, since
acrylamide formation on heating foods is obviously a widespread
phenomenon. Then, in summer 2002, in Geneva, an expert consultation
took place which had been convened jointly by the Food and
Agriculture Organization of the United Nations (FAO) and the World
Health Organization (WHO) (WHO, FAO/WHO Consultation on the Health
Implications of Acrylamide in Food (Geneva, 25-27 Jun. 2002).
[0003] The expert consultation discussed the following as essential
end points of the toxicological effects of acrylamide:
neurotoxicity, reproductive toxicity, mutagenicity and
carcinogenicity.
[0004] In particular, the expert consultation started from the
position that the genotoxic potential of acrylamide and its
metabolic product glycidamide plays an important role. In vivo,
acrylamide is genotoxic in somatic cells and in germ cells. It can
therefore cause inheritable damage at the level of the genes and
also the chromosomes. As is known, one of its metabolic products is
glycidamide, a chemically reactive epoxide, which can react
directly with DNA and form adducts. It has been stressed that
genotoxic mechanisms play the important role in the carcinogenicity
of acrylamide.
[0005] The expert consultation assessed the available data from
studies on laboratory animals. The consultation stressed especially
the importance of genotoxic mechanisms of carcinogenesis and was of
the opinion that to date, scarcely any evidence had been provided
for additional alternative mechanisms, for example of a hormonal
nature.
[0006] The international expert consultation describes the
carcinogenic potency of acrylamide in rats as comparable to that of
other carcinogenic substances occurring in certain foods, in part
depending on preparation, for example benzopyrene. However,
acrylamide is the said to occur at higher contents than all other
carcinogenic substances found to date in foods. For humans, the
relative potency of carcinogenic substances in foods is unknown.
The data from epidemiological studies of workers exposed at work
are of lesser importance, since they are not all suitable for
determining small changes in the risk of cancer. Overall, the
expert consultation assessed the presence of acrylamide in foods as
causing concern.
[0007] On the bases of the available data, the WHO/FAO expert
consultation came to the conclusion that foods make an important
contribution to exposure of consumers.
[0008] Acrylamide is formed when certain foods are prepared at
relatively high temperatures. In addition to the high temperature,
the duration of exposure to high temperatures plays a part. The
international expert consultation did not find any other reliable
evidence for the mechanism formation. The mechanisms of acrylamide
formation, according to the expert consultation, are still not
understood.
[0009] Under certain experimental conditions, acrylamide appears to
form in vitro in the reaction of amino acids, in particular
asparagine (Mottram et al., Nature 419, (2002), 448; Stadler et
al., Nature 419, (2002), 449) with sugars, for example fructose,
galactose, lactose or sucrose (Stadler et al., Nature 419, (2002),
449).
[0010] The causes of the variability in acrylamide contents in
heat-treated foods are not yet sufficiently understood (WHO,
FAO/WHO Consultation on the Health Implications of Acrylamide in
Food (Geneva, 25-27 Jun. 2002)). The international expert
consultation convened by the FAO and the WHO recommended study of
the relationship between processing conditions of foods and the
formation of acrylamide, and also the optimization of processing
conditions with the aim of minimizing acrylamide contents.
[0011] Processes for minimizing acrylamide contents in heat-treated
foods have not yet been described to date in the prior art and are
urgently required. The object therefore underlying the present
invention is to provide processes which permit the production of
heat-treated foods which, compared with conventional heat-treated
foods, have a reduced acrylamide content.
[0012] This object is achieved by the provision of the embodiments
described in the patent claims.
[0013] The present invention therefore relates to a process for
reducing the acrylamide content of heat-treated foods compared with
corresponding conventional heat-treated foods comprising [0014] a)
providing or selecting plant material which, compared with
corresponding conventional plant material, has a reduced content of
soluble sugars and/or amino acids; [0015] b) processing the said
plant material to give a food; and [0016] c) heat-treating the food
produced in process step b).
[0017] Acrylamide (CAS number 79-06-1), which is also called
2-propenamide, vinylamide or ethylenecarboxamide, is a solid
colourless at room temperature which is very soluble in water but
insoluble in heptane. The term "reduction in acrylamide content",
in the context of the present invention, is to be taken to mean the
reduction of the acrylamide content by at least 15%, in particular
by at least 30%, preferably by at least 50%, 75% and particularly
preferably by at least 90%, compared with the acrylamide content of
corresponding conventional heat-treated foods.
[0018] Methods of determining the acrylamide content of foods have
been described, for example, in Tareke et al. (J. Agric. Food Chem.
50, (2002), 4998-5006). The acrylamide was determined
quantitatively by GC/MS after derivatization (e.g. to form the
dibromo product), or by LC/MS-MS, preferably by LC/MS-MS as
described by Tareke et al. (J. Agric. Food Chem. 50, (2002),
4998-5006). Derivatization to form the dibromo product can be
carried out, for example, as in EPA method 8032A
(http://www.epa.gov/epaoswer/hazwaste/test/pdfs/8032a.pdf, December
1996 version, "Acrylamide by gas chromatography") of the US
Environmental Protection Agency (=EPA). In the context of the
present invention the derivatization is preferably carried out
according to EPA method 8032A.
[0019] The term "food", in the context of the present invention, is
to be taken to mean any food which contains plant material. The
term comprises, in particular, preliminary stages, for example
dough mixtures, potato slices, potato strips, granules and maize
grains which are suitable for producing "heat-treated foods". The
preliminary stages, in particular potato slices, for producing the
heat-treated foods may also be present in the precooked or blanched
form or frozen form.
[0020] The term "heat-treated food", in the context of the present
invention, is to be taken to mean any food which has been exposed
to temperatures of >100.degree. C., preferably of 110.degree. C.
to 230.degree. C., in particular 120.degree. C.-200.degree. C.,
preferably of 150.degree. C.-170.degree. C., particularly
preferable 150.degree. C.-180.degree. C. The term "heat treatment",
in the context of the present invention, is to be taken to mean any
treatment which, under standard pressure conditions, leads to
temperatures of above 100.degree. C., in particular it is to be
taken to mean deep-fat frying, grilling, frying, roasting,
extruding, backing or microwave heating, autoclaving or
parfrying.
[0021] The heat treatment time can differ depending on the food.
The absolute acrylamide contents always increase with the heat
treatment time. With the aid of the present invention it is now
possible to lower the acrylamide content of a food which has been
heat-treated at a defined temperature for a defined time by a
defined method, compared with conventional heat-treated foods.
[0022] In the context of the present invention, especially with
regard to potato chips and crisps, the heat treatment, when this is
a deep-fat frying process, is carried out for 10 seconds to 8
minutes, preferably for 2 to 5 minutes, particularly preferably for
2 to 3 minutes. If the heat treatment is a baking process, the heat
treatment is carried out, in the context of the present invention,
for one to 120 minutes, preferably for 5 to 30 minutes.
[0023] In the context of the present invention, especially with
regard to the production of partially fried (parfried) potato
chips, the heat treatment of the potato strips is a deep-fat
parfrying process in oil, which can be carried out for 30 seconds
to 600 seconds, preferably for 60 seconds to 360 seconds and/or the
parfrying temperature may range from 120.degree. C. to 200.degree.
C., preferably from 130.degree. C. to 170.degree. C. In general,
the parfrying time should be sufficient to reduce the moisture of
the potato slices to a moisture content of less than 75% by weight.
Parfried and frozen potato strips intended for finish preparation
by frying are typically parfried to a moisture content of 60-70% by
weight. Frozen potato strips designed for finish preparation by
oven heating are generally parfried to a lower moisture content of
less than 60%, preferably of 40%-55%, and more preferably of of
44%-50% by weight.
[0024] The actual time required for the parfrying step is
determined by several factors, including the specific oil
temperature, dimensions and temperature of the potato slice, the
batch size, volume of the frying kettle and initial moisture
content of the potato slices.
[0025] Preferably, the moisture content is determined as described
in International Patent Application WO 97/40707 A1 on page 14.
[0026] Examples of such "heat-treated foods" are potato crisps
(synonymous to this English term is the American term "potato
chip"), (potato) chips (synonymous to this English term is the
American term "French fry"), parfried potato chips (which can be
optionally frozen after the heat treatment), mashed potato,
biscuits, crackers, crisp bread, breakfast cereals, maize crisps
(tacos), popcorn, bread crisps, wafers, salt sticks, coffee, bread,
rolls, cakes, rice crisps, pizza and toast, in addition tortillas,
croquettes, wedges, potato sticks, twisters, bread coatings for
meat, fish and vegetables, bread coatings for nuts, tortilla chips,
bread and various baked goods and cereal formulations as well as
pre-cooked meals, especially baby food.
[0027] In the context of the present invention the English terms
"potato crisp" and "potato chips" are used instead of the
synonymous American terms "potato chips" and "French fry".
[0028] The term "conventional heat-treated food", in the context of
the present invention, is to be taken to mean a food which has been
produced from conventional plant material. The term "corresponding
conventional heat-treated food", in the context of the present
invention, preferably relates to a heat-treated food which has been
produced from conventional plant material which has been processed
and heat-treated in the same manner as the plant material to be
used according to the invention which, compared with corresponding
conventional plant material, however, has a reduced content of
soluble sugars and/or amino acids, owing to a genetic
modification.
[0029] The term "plant material", in the context of the present
invention, is to be taken to mean any material which consists of
plants or comprises parts of plants. Preferably, the said parts of
plants are harvested products of plants, for example tubers,
fruits, seeds, onions, leaves and roots. The plant material can
originate from any desired plant species, that is to say both
monocotyledonous and also dicotyledonous plants. Preferably this is
plant material from agricultural farmed plants, that is to say from
plants which are cultivated by humans for purposes of nutrition or
for technical, in particular, industrial, purposes. Particular
preference is given to plant material from starchy plants (for
example wheat, barley, oats, rye, potatoes, maize, rice, peas,
manioc), in particular from potato plants.
[0030] The term "conventional plant material", in the context of
the present invention, is to be taken to mean, in particular, plant
material of corresponding non-genetically-modified plants, that is
to say of plants which do not have a genetic modification which
leads to a reduction in the content of soluble sugars, in
particular glucose and/or fructose, and/or to a reduction in the
content of amino acids, in particular asparagine, compared with
corresponding wild type plants. Conventional plant material, in the
context of the present invention, however, can also originate from
genetically modified plants which have been genetically modified in
another aspect, but where the genetic modification does not lead to
a reduction in the content of soluble sugars, in particular glucose
and/or fructose, and/or to a reduction in the content of amino
acids, in particular asparagine, compared with corresponding wild
type plants.
[0031] The term "genetic modification" is defined hereinafter.
[0032] The term "soluble sugars", in the context of the present
invention, is to be taken to mean any water-soluble sugars
occurring in plant material, preferably the soluble sugars are
hexoses, preferably reducing sugars, in particular fructose and/or
glucose.
[0033] The term "reducing the content of soluble sugars" or
"reduced content of soluble sugars", in the context of the present
invention, is to be taken to mean reducing the content of soluble
sugars, preferably to mean reducing the content of soluble sugars
of the plant material, in particular fructose and/or glucose, by at
least 10%, in particular by at least 15%, preferably by at least
20%, and particularly preferably by at least 40%, in particular by
50%-95%, preferably by 60%-90% compared with the content of soluble
sugars, in particular fructose and/or glucose, of corresponding
conventional heat-treated foods or of corresponding conventional
plant material.
[0034] The term "amino acid", in the context of the present
invention, is to be taken to mean any amino acid occurring in plant
material, preferably alanine, arginine, aspartic acid, cysteine,
glutamine, methionine, threonine and valine, more preferably
asparagine.
[0035] The term "reducing the content of amino acids" or "reduced
content of amino acids", in the context of the present invention,
is to be taken to mean reducing the content of amino acids,
preferably to mean reducing the content of amino acids of the plant
material, in particular asparagine, by at least 10%, in particular
by at least 15%, preferably by at least 20%, and particularly
preferably by at least 40%, compared with the content of amino
acids, in particular asparagine, of corresponding conventional
heat-treated foods or of corresponding conventional plant
material.
[0036] The causes of the variability of acrylamide content in
heat-treated foods are not yet adequately understood (WHO, FAO/WHO
Consultation on the Health Implications of Acrylamide in Food
(Geneva, 25-27 June 2002), so that to date no processes for
minimizing acrylamide contents of heat-treated foods have been
described. In particular, no processes were described which have
the selection of particular plant materials as their basis.
[0037] It has now surprisingly been found that the choice of the
starting plant material which is used to produce heat-treated foods
has a critical effect on the acrylamide content of such foods. The
invention teaches for the first time that the use of plant material
which, compared with corresponding conventional plant material, has
a reduced content of soluble sugars and/or amino acids permits the
production of foods which, after heat treatment, have a lower
acrylamide content than in the case of the use of plant material
having conventional contents of soluble sugars and/or amino acids.
The present invention therefore teaches, to avoid the formation of
acrylamide in heat-treated foods, to use plant material which has a
comparatively low content of soluble sugars and/or amino acids.
[0038] Methods of determining the content of sugars, in particular
fructose and glucose, in plant material are known to those skilled
in the art and are described, for example, in Muller-Rober et al.
(Mol. Gen. Genet. 224, (1990), 136-146) and also in the text which
follows. In context with the present invention, the determination
of the content of glucose, fructose and/or sucrose is preferably
performed as described below ("Determination of glucose, fructose
and sucrose").
[0039] Methods of determining the content of amino acids, in
particular asparagine, in plant material are known to those skilled
in the art and are described, for example, in Cohen, Meys, Tarvin
(1988), The pico-tag method: A Manual of advanced techniques for
amino acid analysis, Millipore Corporation, Milford, Mass., USA.
Preferred is the method described by Roessner et al. (Plant
Physiology 127, (2001), 749-764).
[0040] In a further embodiment of the inventive process, the plant
material used is characterized in that it is genetically modified,
the genetic modification leading to a reduction in the content of
soluble sugars, in particular glucose and/or fructose, compared
with corresponding conventional plant material of wild type
plants.
[0041] The "genetic modification", in the context of the present
invention, can be any genetic modification which leads to a
reduction in the content of soluble sugars, compared with
corresponding conventional plant material of wild type plants.
[0042] In the context of the present invention, the genetic
modification can be caused by mutagenesis of one or more genes. The
type of mutation is not critical for this, provided that it leads
to a reduction in the content of soluble sugars compared with
corresponding conventional plant material of wild type plants.
[0043] The term "mutagenesis", in the context of the present
invention, is to be taken to mean any type of mutation, for example
deletions, point mutations (nucleotide replacements), insertions,
inversions, gene conversions or chromosome translocations.
[0044] The mutation can be caused by the use of chemical agents or
high-energy radiation (for example X-ray, neutron, gamma or UV
radiation).
[0045] Agents which can be used for causing chemically induced
mutations and the mutations resulting thereby by reaction of the
corresponding mutagens are described, for example, in Ehrenberg and
Husain, 1981, (Mutation Research 86, 1-113), Muller, 1972
(Biologisches Zentralblatt 91 (1), 31-48). The generation of rice
mutants using gamma rays, ethyl methanesulphonate (EMS),
N-methyl-N-nitrourea or sodium azide (NaN.sub.3) is described, for
example, in Jauhar and Siddiq (1999, Indian Journal of Genetics, 59
(1), 23-28), in Rao (1977, Cytologica 42, 443-450), Gupta and
Sharma (1990, Oryza 27, 217-219) and Satoh and Omura (1981,
Japanese Journal of Breeding 31 (3), 316-326). The generation of
wheat mutants using NaN.sub.3 and maleic hydrazide
(1,2-dihydropyridazine-3,6-dione) is described, by way of example,
in Arora et al. (1992, Annals of Biology 8 (1), 65-69). A review of
the production of wheat mutants using various types of
higher-energy radiation and chemical agents is given in
Scarascia-Mugnozza et al. (1993, Mutation Breeding Review 10,
1-28). Svec et al. (1998, Cereal Research Communications 26 (4),
391-396) describe the use of N-ethyl-N-nitrourea for generating
mutants in triticale. The use of MMS and gamma radiation for
generating millet mutants is described in Shashidhara et al. (1990,
Journal of Maharashtra Agricultural Universities 15 (1),
20-23).
[0046] The production of mutants in plant species which principally
reproduce vegetatively has been described, for example, for
potatoes which produce a modified starch (Hovenkamp-Hermelink et
al. (1987, Theoretical and Applied Genetics 75, 217-221), and for
mint having an increased oil yield and modified oil quality
(Dwivedi et al., 2000, Journal of Medicinal and Aromatic Plant
Sciences 22, 460-463).
[0047] All of these methods are suitable in principle for providing
plant material which, owing to a genetic modification, has a
reduced content of soluble sugars compared with corresponding
conventional plant material of wild type plants and is therefore
suitable for use in the inventive process.
[0048] Mutations in the appropriate genes can be discovered using
methods known to those skilled in the art. In particular, those
which can be employed for this purpose are analyses based on
hybridization with probes (Southern Blot), amplification by means
of the polymerase chain reaction (PCR), sequencing relevant genomic
sequences, and searching for individual nucleotide replacements. A
method for identifying mutations on the basis of hybridization
patterns is, for example, the search for restriction fragment
length polymorphism (RFLP) (Nam et al., 1989, The Plant Cell 1,
699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750).
A method based on PCR is, for example, the analysis of amplified
fragment length polymorphism (AFLP) (Castiglioni et al., 1998,
Genetics 149, 2039-2056; Meksem et al., 2001, Molecular Genetics
and Genomics 265, 207-214; Meyer et al., 1998, Molecular and
General Genetics 259, 150-160). The use of amplified fragments
cleaved by restriction endonucleases (Cleaved Amplified Polymorphic
Sequences, CAPS) can also be used for identifying mutations
(Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis
et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et al.,
1996, The Plant Journal 9 (5), 745-753). Methods for determining
SNPs (Single Nucleotide Polymorphisms) have been described, inter
alia, by Qi et al. (2001, Nucleic Acids Research 29 (22), el16)
Drenkard et al. (2000, Plant Physiology 124, 1483-1492) and Cho et
al. (1999, Nature Genetics 23, 203-207). In particular, methods are
suitable which permit many plants to be studied within a short time
for mutations in certain genes. Such a method, called TILLING
(Targeting Induced Local Lesions IN Genomes), has been described,
by way of example, by McCallum et al. (2000, Plant Physiology 123,
439-442).
[0049] The use of all of these methods is suitable in principle for
identifying genetically modified plant material which is suitable
for use in the inventive process.
[0050] In addition, the genetically modified plant material which
can be used in the context of the present invention can be produced
by genetic engineering methods (antisense, cosuppression
technology, ribozymes, in-vivo mutagenesis, RNAi-Technology,
etc.).
[0051] In a further embodiment of the inventive process, which is
particularly preferred, the genetic modification leads to a
reduction in the activity of one or more endogenous R1 proteins
occurring in the plant cell, compared with corresponding plant
cells of wild type plants which have not been genetically
modified.
[0052] The term "R1 protein", in the context of the present
invention, is to be taken to mean proteins which have been
described, for example, in Lorberth et al. (Nature Biotech. 16,
(1998), 473-477), Ritte et al., (PNAS 99, (2002), 7166-7171) and in
the international patent applications WO98/27212, WO00/77229,
WO00/28052 and have the characteristics below. Important
characteristics of R1 proteins are i) their amino acid sequence
(see, for example, GenBank Acc. No. A61831, Y09533); ii) their
localization in the plastids of plant cells; iii) their ability to
affect the degree of phosphorylation of the starch in plants.
Further, the term "R1 protein" refers to a protein catalysing the
phosphorylation of starch in a dikinase-type reaction in which
three substrates, an .alpha.-polyglucan, ATP and H.sub.2O are
converted into three products, an .alpha.-polyglucan-P, AMP and
orthophosphate (Rifte et al., PNAS Vol. 99 No. 10, (2002),
7166-7171). A synonym, which is used in the more recent literature
for the term "R1 protein", is the term "GWD protein" which is the
abbreviation for "alpha-glucan water dikinase" (Blennow et al.,
Trends in Plant Science Vol. 7 No. 10 (2002), 445-450). Therefore,
with respect to the present invention, the term "R1 protein"
comprises also "GWD proteins".
[0053] For example, inhibiting the R1 gene coding for an R1 protein
from potatoes leads, in transgenic potato plants, to a reduction in
the phosphate content of the starch which can be isolated from the
potato tubers. Moreover, Lorberth et al. show that the R1 protein
from Solanum tuberosum is able to phosphorylate bacterial glycogen
when the corresponding R1 cDNA is expressed in E. coli (Lorberth et
al., Nature Biotech. 16, (1998), 473-477).
[0054] Ritte et al. (Plant J. 21, (2000), 387-391) showed that the
R1 protein from Solanum tuberosum in potato plants reversibly binds
to starch granules, with the strength of the binding to the starch
granule being dependent on the metabolic status of the plant. In
the form bound to starch granules, the protein in potato plants is
principally present in leaves which have been grown in the dark.
After illuminating the leaves the protein, in contrast, is
principally present in the soluble form which is not bound to the
starch granule.
[0055] In the context of the present invention, it is of particular
importance that inhibiting the expression of the potato R1 gene in
transgenic potato plants or their tubers leads to a reduction of
what is termed cold-induced sweetenings (Lorberth et al., Nature
Biotech. 16, (1998), 473-477), that is to say cold-stored potato
tubers, compared with tubers of corresponding wild type plants
which are not genetically modified, have a reduced content of
soluble sugars, in particular fructose and glucose.
[0056] In addition, the potato tubers of these transgenic plants
having reduced R1 gene expression, even immediately after harvest,
or after storage at room temperature, compared with tubers of
corresponding wild type plants which are not genetically modified,
have a reduced content of soluble sugars, in particular fructose
and glucose.
[0057] By means of the present invention, it has been demonstrated
for the first time that deep-fat frying or parfrying of
(cold-stored) potato tubers which originate from plants having
reduced R1 gene expression (Lorberth et al., Nature Biotech. 16,
(1998), 473-477), leads to a markedly reduced acrylamide formation
in the deep-fat-fried products than when corresponding potato
tubers which have not been genetically modified are deep-fat fried.
The reduction in acrylamide formation is surprisingly high.
[0058] In a further embodiment of the inventive process, the
genetic modification leads to a reduction in the activity of one or
more endogenous invertase proteins occurring in the plant cell,
compared with corresponding plant cells of wild type plants which
have not been genetically modified.
[0059] The term "invertase protein", in the context of the present
invention, is to be taken to mean proteins having the enzymatic
activity of an invertase. Invertases catalyze the cleavage of
sucrose into glucose and fructose.
[0060] Preferably, in the context of the present invention, these
are acid invertases, which are also called vacuolar invertases, and
have been described, for example, in Zrenner et al. (Planta 198,
(1996), 246-252).
[0061] Potato plants having decreased invertase activity have been
described, for example, in Zrenner et al. (Planta 198, (1996),
246-252) and in Greiner et al. (Nature Biotechnology 17, (1999),
708-711).
[0062] In the context of the present invention, it is of particular
importance that the reduction in invertase activity in transgenic
potato plants, in particular in those which express a vacuolar
invertase inhibitor from tobacco (Greiner et al., Nature
Biotechnology 17, (1999), 708-711), leads to cold-stored potato
tubers of these transgenic plants having a decreased content of
soluble sugars, in particular fructose and glucose, compared with
tubers of corresponding wild type plants which have not been
genetically modified.
[0063] The term "reduction in activity", in the context of the
present invention, means a reduction compared with corresponding
non genetically-modified cells in the expression of endogenous
genes which code for R1 or invertase proteins and/or a reduction of
the amount of R1 protein or invertase protein in the cells of the
plant material and/or a reduction in the enzymatic activity of the
R1 or invertase proteins in the cells of the plant material.
[0064] The term "reduction in the activity of one or more
endogenous R1 proteins occurring in the plant cell", in the context
of the present invention, is to be taken to mean a reduction in the
expression of one or more endogenous genes which code for R1
proteins, and/or a reduction in the amount of R1 protein in the
cells of the plant material and/or a reduction in the enzymatic
activity of the R1 proteins in the cells of the plant material
compared with corresponding non genetically-modified cells of
wildtype plants.
[0065] The term "reduction in the activity of one or more
endogenous invertase proteins occurring in the plant cell", in the
context of the present invention, is to be taken to mean a
reduction in the expression of one or more endogenous genes which
code for invertase proteins, and/or a reduction in the amount of
invertase protein in the cells of the plant material and/or a
reduction in the enzymatic activity of the invertase proteins in
the cells of the plant material compared with corresponding non
genetically-modified cells.
[0066] The reduction in expression can be determined, for example,
by measuring the amount of transcripts coding for R1 or invertase
protein, for example by Northern blot analysis or RT-PCR. A
reduction preferably means a reduction in the amount of transcripts
compared with the corresponding non genetically-modified cells by
at least 50%, in particular by at least 70%, preferably by at least
85%, and particularly preferably by at least 95%.
[0067] The reduction in the amount of R1 or invertase proteins
which results in a reduced activity of these proteins in the plant
cells in question can be determined, for example, by immunological
methods, such as Western blot analysis, ELISA (Enzyme Linked Immuno
Sorbent Assay) or RIA (Radio Immune Assay). A reduction preferably
means a reduction in the amount of R1 or invertase protein compared
with the corresponding non genetically-modified cells by at least
50%, in particular by at least 70%, preferably by at least 85%, and
particularly preferably by at least 95%.
[0068] The reduction in the enzymatic activity of the R1 protein
can be determined on the basis of an enzymatic assay described by
Ritte et al. (PNAS 99, (2002), 7166-7171).
[0069] The reduction in enzymatic activity of the invertase protein
can be determined by the method described by Greiner et al. (Nature
Biotechnology 17, (1999), 708).
[0070] A reduction in the enzymatic activity of the R1 or invertase
protein preferably means a reduction in activity compared with
corresponding non genetically-modified cells by at least 50%, in
particular by at least 60%, and preferably by at least 70%.
[0071] A reduction in the enzymatic activity of the R1 protein
preferably means a reduction in activity of R1 compared with R1
activity of corresponding non genetically-modified cells by at
least 50%, in particular by at least 60%, and preferably by at
least 70%.
[0072] A reduction in the enzymatic activity of the invertase
protein preferably means a reduction in activity of the invertase
protein compared with invertase activity of corresponding non
genetically-modified cells by at least 50%, in particular by at
least 60%, and preferably by at least 70%.
[0073] In a further embodiment of the inventive process, the
genetic modification is the introduction of one or more foreign
nucleic acid molecules, the presence and/or expression of which
leads to the reduction in the activity of one or more endogenous R1
proteins occurring in the plant cell compared with corresponding
plant cells of wild type plants which have not been genetically
modified.
[0074] In a further embodiment of the inventive process, the
genetic modification is the introduction of one or more foreign
nucleic acid molecules, the presence and/or expression of which
leads to the reduction in the activity of one or more endogenous
invertase proteins occurring in the plant cell compared with
corresponding plant cells of wild type plants which have not been
genetically modified.
[0075] The term "foreign nucleic acid molecule" or "foreign nucleic
acid molecules", in the context of the present invention, is to be
taken to mean a molecule which either does not occur naturally in
corresponding plant cells, or which does not occur naturally in the
plant cells in the specific spatial arrangement or which is
localized at a site in the genome of the plant cell at which it
does not naturally occur. Preferably, the foreign nucleic acid
molecule is a recombinant molecule which consists of various
elements, the combination or specific spatial arrangement of which
does not occur naturally in plant cells.
[0076] In a further preferred embodiment of the inventive process,
the foreign nucleic acid molecule is selected from the group
consisting of [0077] (a) DNA molecules which code for at least one
antisense RNA causing a reduction in expression of endogenous genes
which code for R1 proteins; [0078] (b) DNA molecules which, via a
cosuppression effect, lead to reduction of the expression of
endogenous genes coding for R1 proteins; [0079] (c) DNA molecules
which code for at least one ribozyme which cleaves in a specific
manner transcripts of endogenous genes coding for R1 proteins;
[0080] (d) nucleic acid molecules which are introduced by means of
in vivo mutagenesis and lead to a mutation or insertion of a
heterologous sequence in genes coding for endogenous R1 proteins,
the mutation or insertion causing a reduction in the expression of
the said genes or the synthesis of inactive R1 proteins; [0081] (e)
DNA molecules which simultaneously code for at least one antisense
RNA and at least one sense RNA, the said antisense RNA and the said
sense RNA forming a double-stranded RNA molecule which causes a
reduction in the expression of endogenous genes coding for R1
proteins; [0082] (f) DNA molecules which contain transposons, the
integration of the transposon sequences leading to a mutation or an
insertion in endogenous genes coding for R1 proteins which causes a
reduction in the expression of the said genes or the synthesis of
inactive R1 proteins; and [0083] (g) T-DNA molecules which, via
insertion in endogenous genes coding for R1 protein cause a
reduction in the expression of genes coding for R1 protein or the
synthesis of inactive R1 proteins.
[0084] In a further preferred embodiment of the inventive process,
the foreign nucleic acid molecule is selected from the group
consisting of [0085] (a) DNA molecules which code for an invertase
inhibitor. [0086] (b) DNA molecules which code for at least one
antisense RNA which causes a reduction in expression of endogenous
genes coding for invertase proteins; [0087] (c) DNA molecules
which, via a cosuppression effect, lead to reduction of the
expression of endogenous genes coding for invertase proteins;
[0088] (d) DNA molecules which code for at least one ribozyme which
cleaves in a specific manner transcripts of endogenous genes coding
for invertase proteins; [0089] (e) nucleic acid molecules which are
introduced by means of in-vivo mutagenesis and which lead to a
mutation or an insertion of a heterologous sequence in endogenous
genes coding for invertase proteins, the mutation or insertion
causing a reduction in the expression of the said genes or the
synthesis of inactive invertase proteins; [0090] (f) DNA molecules
which simultaneously code for at least one antisense RNA and at
least one sense RNA, the said antisense RNA and the said sense RNA
forming a double-stranded RNA molecule which causes a reduction in
the expression of endogenous genes coding for invertase proteins;
[0091] (g) DNA molecules which contain transposons, the integration
of the transposon sequences leading to a mutation or an insertion
in endogenous genes coding for invertase proteins, which causes a
reduction in the expression of the said genes or the synthesis of
inactive invertase proteins; and [0092] (h) T-DNA molecules which,
via insertion in endogenous genes coding for invertase protein,
cause a reduction in the expression of genes coding for invertase
protein, or the synthesis of inactive invertase proteins.
[0093] To inhibit the gene expression by means of antisense or
cosuppression techniques, for example, a DNA molecule can be used
which comprises the entire sequence coding for an R1 protein or
invertase protein and possibly existing flanking sequences, and
also DNA molecules which comprise only part of the coding sequence,
with these parts needing to be long enough to cause an antisense
effect or cosuppression effect in the cells. Suitable sequences are
generally sequences up to a minimum length of 15 bp, preferably a
minimum length of 21 bp, preferably a length of 100-500 bp, and for
an efficient antisense or cosuppression inhibition, particular
preference is given to sequences having a length over 500 bp. These
statements apply correspondingly to the inhibition of BE I gene
expression.
[0094] For antisense or cosuppression approaches, it is also
suitable to use DNA sequences which have a high degree of homology
to the endogenous sequences in the plant cell which code for an R1
protein or invertase protein. The minimum homology should be
greater than approximately 65%. The use of sequences having
homologies of at least 90%, in particular between 95 and 100%, is
to be preferred. These statements apply correspondingly to the
inhibition of BE I gene expression.
[0095] In addition, to achieve an antisense or cosuppression
effect, the use of introns is also conceivable, that is to say
non-coding regions of genes which code for an R1 protein or
invertase protein.
[0096] The use of intron sequences for inhibiting gene expression
of genes which code for proteins of starch biosynthesis has been
described, for example, in the international patent applications
WO97/04112, WO97/04113, WO98/37213, WO98/37214. These statements
apply correspondingly to the inhibition of BE I gene
expression.
[0097] A person skilled in the art is familiar with how to achieve
an antisense and cosuppression effect. The process of cosuppression
inhibition has been described, for example, in Jorgensen (Trends
Biotechnol. 8 (1990), 340-344), Niebel et al., (Curr. Top.
Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top.
Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret
(Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al., (Mol.
Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet.
243 (1994), 613-621).
[0098] The expression of ribozymes for reducing activity of certain
enzymes in cells is also known to those skilled in the art and is
described, for example, in EP-B1 0321201. The expression of
ribozymes in plant cells has been described, for example, in Feyter
et al. (Mol. Gen. Genet. 250, (1996), 329-338).
[0099] In addition, the reduction of the R1 or invertase activity
in the plant cells of the plant material can also be achieved by
"in-vivo mutagenesis", in which, by transformation of cells, a
hybrid RNA-DNA oligonucleotide ("chimeroplast") is introduced into
cells (Kipp, P. B. et al., Poster Session at the 5.sup.th
International Congress of Plant Molecular Biology, 21.-27.
September 1997, Singapore; R. A. Dixon and C. J. Arntzen, Meeting
report on "Metabolic Engineering in Transgenic Plants", Keystone
Symposia, Copper Mountain, Colo., USA, TIBTECH 15, (1997), 441-447;
international patent application WO 9515972; Kren et al.,
Hepatology 25, (1997), 1462-1468; Cole-Strauss et al., Science 273,
(1996), 1386-1389; Beetham et al., (1999), PNAS 96, 8774-8778).
[0100] A part of the DNA component of the RNA-DNA oligonucleotide
is homologous to a nucleic acid sequence of an endogenous R1 or
invertase gene, but, compared therewith, has a mutation or contains
a heterologous region which is enclosed by the homologous
regions.
[0101] By base-pairing the homologous regions of the RNA-DNA
oligonucleotide and of the endogenous nucleic acid molecule,
followed by homologous recombination, the mutation or heterologous
region present in the DNA component of the RNA-DNA oligonucleotide
can be transferred into the genome of a plant cell. This leads to a
reduction in activity of one or more R1 or invertase proteins.
These statements apply correspondingly to the inhibition of BE I
gene expression.
[0102] In addition, the R1 or invertase activity can also be
reduced in the plant cells by the simultaneous expression of sense
and antisense RNA molecules of the respective target gene to be
repressed, preferably of the R1 or invertase gene.
[0103] This can be achieved, for example, by using chimeric
constructs which contain inverted repeats of the respective target
gene or parts of the target gene. In this case the chimeric
constructs code for sense and antisense RNA molecules of the
respective target gene. Sense and antisense RNA are synthesized in
planta simultaneously as one RNA molecule, with sense and antisense
RNA being separated from one another by a spacer and able to form a
double-stranded RNA molecule. This technology is also called "RNAi
technology".
[0104] It has been shown that introducing inverted repeat DNA
constructs into the genome of plants is a highly efficient method
for repressing the genes corresponding to the inverted repeat DNA
constructs (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95,
(1998), 13959-13964; Wang and Waterhouse, Plant Mol. Biol. 43,
(2000), 67-82; Singh et al., Biochemical Society Transactions Vol.
28 part 6 (2000), 925-927; Liu et al., Biochemical Society
Transactions Vol. 28 part 6 (2000), 927-929); Smith et al., (Nature
407, (2000), 319-320; international patent application WO99/53050
A1). Sense and antisense sequences of the target gene or the target
genes can also be expressed separately from one another by means of
the same or different promoters (Nap, J-P et al, 6.sup.th
International Congress of Plant Molecular Biology, Quebec, 18-24
Jun., 2000; Poster S7-27, Presentation Session S7). These
statements apply correspondingly to the inhibition of BE I gene
expression.
[0105] The reduction in R1 or invertase activity in the plant cells
of the plant material can thus also be achieved by producing
double-stranded RNA molecules of R1 or invertase genes. Preferably,
for this purpose, inverted repeats of DNA molecules of R1 or
invertase genes or cDNAs are introduced into the genome of plants,
the DNA molecules to be transcribed (R1 or invertase genes or cDNAs
or fragments of these genes or cDNAs) being under the control of a
promoter which controls the expression of the said DNA molecules.
These statements apply correspondingly to the inhibition of BE I
gene expression.
[0106] Furthermore it is known that the formation of
double-stranded RNA molecules of promoter DNA molecules in plants
in trans can lead to a methylation and a transcriptional
inactivation of homologous copies of these promoters which are to
be termed hereinafter target promoters (Mette et al., EMBO J. 19,
(2000), 5194-5201).
[0107] Via the inactivation of the target promoter it is thus
possible to reduce the gene expression of a certain target gene
(for example R1 or invertase gene) which exists naturally under the
control of this target promoter.
[0108] That is to say the DNA molecules which comprise the target
promoters of the genes to be repressed (target genes) are in this
case, in contrast to the original function of promoters in plants,
used not as control elements for the expression of genes or cDNAs,
but are themselves used as transcribable DNA molecules.
[0109] To produce the double-stranded target promoter RNA molecules
in planta which can be present there as RNA hairpin molecules,
preferably, constructs are used which contain inverted repeats of
the target promoter DNA molecules, the target promoter DNA
molecules being under the control of a promoter which controls the
gene expression of the said target promoter DNA molecules. These
constructs are then introduced into the genome of plants. The
expression of the "inverted repeats" of the said target promoter
DNA molecules leads in planta to the formation of double-stranded
target promoter RNA molecules (Mette et al., EMBO J. 19, (2000),
5194-5201). By this means the target promoter can be inactivated.
The reduction in R1 or invertase activity in the plant cells can
thus also be achieved by producing double-stranded RNA molecules of
promoter sequences of R1 or invertase genes. Preferably, for this
purpose, inverted repeats of promoter DNA molecules of R1 or
invertase promoters are introduced into the genome of plants, the
target promoter DNA molecules to be transcribed (R1 or invertase
promoter) being under the control of a promoter which controls the
expression of the said target promoter DNA molecules. These
statements apply correspondingly to the inhibition of BE I gene
expression.
[0110] In a further embodiment of the present invention, the
foreign nucleic acid molecule is inserted transposons or what is
called transfer DNA (T-DNA) into a gene coding for an R1 or
invertase protein, the activity of the said proteins being reduced
as a result in the relevant cell of the plant material. These
statements apply correspondingly to the inhibition of BE I gene
expression.
[0111] In principle, the plant material suitable for the inventive
process can be produced not only using homologous, but also
heterologous, transposons, the use of homologous transposons also
being taken to mean those which are already naturally present in
the plant genome. These statements apply correspondingly to the
inhibition of BE I gene expression.
[0112] Modifying gene expression by means of transposons is known
to those skilled in the art. A review of the use of endogenous and
heterologous transposons as tools in plant biotechnology is given
in Ramachandran and Sundaresan (2001, Plant Physiology and
Biochemistry 39, 234-252). The possibility of identifying mutants
in which specific genes have been inactivated by transposon
insertion mutagenesis is described in a review by Maes et al.
(1999, Trends in Plant Science 4 (3), 90-96). Producing rice
mutants with the aid of endogenous transposons is described by
Hirochika (2001, Current Opinion in Plant Biology 4, 118-122). The
identification of maize genes using endogenous retrotransposons is
reported, for example, by Hanley et al. (2000, The Plant Journal 22
(4), 557-566). The possibility of producing mutants using
retrotransposons and methods for identifying mutants are described
by Kumar and Hirochika (2001, Trends in Plant Science 6 (3),
127-134). The activity of heterologous transposons in different
species has been described not only for dicotyledonous but also for
monocotyledonous plants: e.g. for rice (Greco et al., 2001, Plant
Physiology 125, 1175-1177; Liu et al., 1999, Molecular and General
Genetics 262, 413-420; Hiroyuki et al., 1999, The Plant Journal 19
(5), 605-613; Jeon and Gynheung, 2001, Plant Science 161, 211-219),
barley (2000, Koprek et al., The Plant Journal 24 (2), 253-263)
Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717,
Schmidt and Willmitzer, 1989, Molecular and General Genetics 220,
17-24; Altmann et al., 1992, Theoretical and Applied Genetics 84,
371-383; Tissier et al., 1999, The Plant Cell 11, 1841-1852),
tomato (Belzile and Yoder, 1992, The Plant Journal 2 (2), 173-179)
and potatoes (Frey et al., 1989, Molecular and General Genetics
217, 172-177; Knapp et al., 1988, Molecular and General Genetics
213, 285-290).
[0113] T-DNA insertion mutagenesis is based on the fact that
certain sections (T-DNA) of Ti-plasmids from Agrobacterium can
integrate into the genome of plant cells. The site of integration
in the plant chromosome is not fixed here, that can be at any
desired position. If the T-DNA integrates into a section of the
chromosome representing a gene function, this can lead to a
modification of gene expression and thus also to a change in the
activity of a protein coded for by the gene in question. In
particular, the integration of a T-DNA into the coding region of a
protein frequently leads to the corresponding protein no longer
being able to be synthesized by the cell in question, or not in
active form. The use of T-DNA insertions for producing mutants is
described, for example, for Arabidopsis thaliana (Krysan et al.,
1999, The Plant Cell 11, 2283-2290; Atipiroz-Leehan and Feldmann,
1997, Trends in genetics 13 (4), 152-156; Parinov and Sundaresan,
2000, Current Opinion in Biotechnology 11, 157-161) and rice (Jeon
and An, 2001, Plant Science 161, 211-219; Jeon et al., 2000, The
Plant Journal 22 (6), 561-570). Methods for identifying mutants
which have been produced using T-DNA insertion mutagenesis are
described, inter alia, by Young et al., (2001, Plant Physiology
125, 513-518), Parinov et al. (1999, The Plant cell 11, 2263-2270),
Thorneycroft et al. (2001, Journal of Experimental Botany 52,
1593-1601), and McKinney et al. (1995, The Plant Journal 8
(4),613-622).
[0114] T-DNA mutagenesis is suitable in principle for producing the
plant material which can be used in the inventive process.
[0115] In a further embodiment of the inventive process, the
genetic modification not only leads to a reduction in the activity
of one or more endogenous R1 proteins occurring in the plant cell,
but also at the same time to a reduction in the activity of one or
more endogenous branching enzymes of isoform I occurring in the
plant cell (branching enzyme I=BEI protein), compared with
corresponding non-genetically-modified plant cells of wild type
plants.
[0116] The term "BEI protein", in the context of the present
invention, is to be taken to mean a branching enzyme (=BE) of
isoform I. Preferably the BEI protein originates from potato
plants.
[0117] The naming of the isoforms here is based on the nomenclature
proposed by Smith-White and Preiss (Smith-White and Preiss, Plant
Mol Biol. Rep. 12, (1994), 67-71, Larsson et al., Plant Mol Biol.
37, (1998), 505-511). This nomenclature starts from the position
that all enzymes which have a higher homology (identity) at the
amino acid level to the BEI protein from maize (GenBank Acc. No.
D11081; Baba et al., Biochem. Biophys. Res. Commun. 181 (1),
(1991), 87-94; Kim et al. Gene 216, (1998), 233-243) than to the
BEII protein from maize (Genbank Acc. No AF072725, U65948), are
called branching enzyme of isoform I, or for short BEI
proteins.
[0118] Nucleic acid molecules coding for "BEI protein" have been
described for numerous plants, for example for maize (Genbank Acc.
No. D 11081, AF 072724), rice (Genbank Acc. No. D11082), peas
(Genbank Acc. No. X80010) and potatoes. Various forms of the BEI
gene and of the BEI protein from potatoes have been described, for
example, in Khoshnoodi et al., Eur. J. Biochem. 242 (1), 148-155
(1996), Genbank Acc. No. Y 08786 and in Kossmann et al., Mol. Gen.
Genet. 230, (1991), 39-44). In potato plants, the BEI gene is
principally expressed in the tubers and scarcely at all in the
leaves (Larsson et al., Plant Mol. Biol. 37, (1998), 505-511).
[0119] Regarding the genetic modification which leads to a
reduction in the R1 activity, the statements made above apply. The
genetic modification which leads to a reduction in the activity of
the BEI protein I (branching enzyme 1), can be the introduction of
one or more foreign nucleic acid molecules, the presence and/or
expression of which leads to the reduction in the activity of one
or more endogenous BEI proteins of isoform I occurring in the plant
cell compared with corresponding non-genetically-modified plant
cells of wild type plants.
[0120] The term "reduction in the activity of one or more
endogenous branching enzymes of isoform I occurring in the plant
cell", in the context of the present invention, is to be taken to
mean a reduction compared with corresponding non
genetically-modified cells in the expression of one or more
endogenous genes which code for BEI proteins, and/or a reduction in
the amount of BEI protein in the cells of the plant material and/or
a reduction in the enzymatic activity of the BEI proteins in the
cells of the plant material.
[0121] The reduction in expression can be determined, for example,
by measuring the amount of transcripts coding for BEI protein, for
example by Northern blot analysis or RT-PCR. A reduction preferably
means a reduction in the amount of transcripts compared with the
corresponding non genetically-modified cells by at least 50%, in
particular by at least 70%, preferably by at least 85%, and
particularly preferably by at least 95%.
[0122] The reduction in the amount of BEI proteins which results in
a reduced activity of this protein in the plant cells in question
can be determined, for example, by immunological methods, such as
Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay)
or RIA (Radio Immune Assay). A reduction preferably means a
reduction in the amount of BEI protein compared with the
corresponding non genetically-modified cells by at least 50%, in
particular by at least 70%, preferably by at least 85%, and
particularly preferably by at least 95%.
[0123] In a further preferred embodiment of the inventive process,
the foreign nucleic acid molecule which leads to the reduction in
activity of one or more endogenous BEI proteins of isoform I
occurring in the plant cell is selected from the group consisting
of [0124] (a) DNA molecules which code for at least one antisense
RNA causing a reduction in the expression of endogenous genes which
code for BEI proteins; [0125] (b) DNA molecules which, via a
cosuppression effect, lead to reduction in the expression of
endogenous genes which code for BEI proteins; [0126] (c) DNA
molecules which code for at least one ribozyme which cleaves in a
specific manner transcripts of endogenous genes coding for BEI
proteins; [0127] (d) nucleic acid molecules which are introduced by
means of in vivo mutagenesis and lead to a mutation or insertion of
a heterologous sequence in genes coding for endogenous BEI
proteins, the mutation or insertion causing a reduction in the
expression of the said genes or the synthesis of inactive BEI
proteins; [0128] (e) DNA molecules which simultaneously code for at
least one antisense RNA and at least one sense RNA, the said
antisense RNA and the said sense RNA forming a double-stranded RNA
molecule which causes a reduction in the expression of endogenous
genes coding for BEI proteins; [0129] (f) DNA molecules which
contain transposons, the integration of the transposon sequences
leading to a mutation or insertion in endogenous genes coding for
BEI proteins which causes a reduction in the expression of the said
genes or the synthesis of inactive BEI proteins; and [0130] (g)
T-DNA molecules which, via insertion in endogenous genes coding for
BEI protein cause a reduction in the expression of genes coding for
BEI protein or the synthesis of inactive BEI proteins.
[0131] In a further embodiment of the inventive process, the plant
material used is characterized in that it is genetically modified,
with the genetic modification leading to a reduction in the content
of amino acids, in particular asparagine, compared with
corresponding conventional plant material from wild type
plants.
[0132] The "genetic modification", in the context of the present
invention, can be any genetic modification which leads to a
reduction in the content of amino acids, in particular of
asparagine, compared with corresponding conventional plant material
from wild type plants.
[0133] With respect to the differing conceivable ways for producing
genetic modifications which lead to a reduction in the amino acid
content, in particular asparagine, the statements made in general
above apply in the context of the genetic modifications which lead
to a reduction in the content of sugars.
[0134] In a further embodiment of the inventive process, the
genetic modification leads to a reduction in the activity of one or
more endogenous asparagine synthetase proteins occurring in the
plant cell compared with corresponding plant cells of wild type
plants which have not been genetically modified.
[0135] An "asparagine synthetase protein", in the context of the
present invention, is to be taken to mean a protein which catalyses
the conversion of aspartate to asparagine with the conversion of
ATP to AMP and pyrophosphate, and of glutamine to glutamate.
Sequence information for asparagine synthetases (asn1) has been
described, for example, in Lam et al. (Plant Physiol. 106(4),
(1994), 1347-1357).
[0136] Plants having decreased asparagine synthetase activity have,
compared with corresponding wild type plants, reduced content of
asparagine (Annual Meeting of the American Society of Plant
Biologists in Madison, Wis., USA, (1998), Molecular and transgenic
studies of asparagine synthetase genes in Arabidopsis thaliana,
Abstract Number 535).
[0137] With respect to the definition of the term "reduction in
activity", the statements made above in connection with the R1 or
invertase protein apply accordingly. The activity of asparagine
synthetase can be determined, for example, by the method described
by Romagni and Dayan (Journal of Agricultural & Food Chemistry
48(5), (2000), 1692-1696).
[0138] In a further embodiment of the inventive process, the
genetic modification is the introduction of one or more foreign
nucleic acid molecules, the presence and/or expression of which
leads to a reduction in the activity of one or more endogenous
asparagine synthetase proteins occurring in the plant cell,
compared with corresponding plant cells from wild type plants which
have not been genetically modified.
[0139] The term "foreign nucleic acid molecule" here has the
meaning already defined above.
[0140] In a further embodiment of the inventive process, the
foreign nucleic acid molecule is selected from the group consisting
of [0141] (a) DNA molecules which code for at least one antisense
RNA which causes a reduction in the expression of endogenous genes
coding for asparagine synthetase proteins; [0142] (b) DNA molecules
which, via a cosuppression effect, lead to a reduction in the
expression of endogenous genes coding for asparagine synthetase
proteins; [0143] (c) DNA molecules which code for at least one
ribozyme which cleaves in a specific manner transcripts of
endogenous genes coding for asparagine synthetase proteins; [0144]
(d) nucleic acid molecules introduced by in-vivo mutagenesis which
lead to a mutation or insertion of a heterologous sequence into
genes coding for endogenous asparagine synthetase protein, the
mutation or insertion causing a reduction in the expression of the
said genes or the synthesis of inactive asparagine synthetase
protein; [0145] (e) DNA molecules which code simultaneously for at
least one antisense RNA and at least one sense RNA, the said
antisense RNA and the said sense RNA forming a double-stranded RNA
molecule which causes a reduction in the expression of endogenous
genes coding for asparagine synthetase proteins; [0146] (f) DNA
molecules which contain transposons, the integration of the
transposon sequences leading to a mutation or an insertion in genes
coding for endogenous asparagine synthetase proteins which causes a
reduction in the expression of the said genes or the synthesis of
inactive asparagine synthetase proteins; and [0147] (g) T-DNA
molecules which, via insertion in genes coding for endogenous
asparagine synthetase protein, cause a reduction in the expression
of genes coding for asparagine synthetase protein or the synthesis
of inactive asparagine synthetase proteins.
[0148] In this connection, the statements already made generally
above in a different context on carrying out the genetic
engineering approaches (antisense, cosuppression and ribozyme
techniques, in-vivo mutagenesis, transposons, T-DNA insertion)
apply accordingly to the genetic modification of asparagine
synthetase activity.
[0149] In a further embodiment of the inventive process, the
genetic modification leads to an increase in activity of an
ADP-glucose pyrophosphorylase protein, compared with corresponding
plant cells of wild type plants which have not been genetically
modified.
[0150] The ADP glucose pyrophosphorylase activity can be
determined, for example, as described in Muller-Rober et al. ( EMBO
J. 11, (1992), 1229-1238).
[0151] An "ADP-glucose pyrophosphorylase protein", in the context
of the present invention, is to be taken to mean a protein which
catalyses the conversion of glucose-1-phoshate and ATP into
ADP-glucose and pyrophosphate.
[0152] In a further embodiment of the inventive process, the
genetic modification is the introduction of one or more foreign
nucleic acid molecules, the presence and/or expression of which
leads to the increase in the activity of one or more ADP-glucose
pyrophosphorylase proteins occurring in the plant cell compared
with corresponding plant cells of wild type plants which have not
been genetically modified.
[0153] The term "foreign nucleic acid molecule" here has the
meaning already defined above.
[0154] Preferably, the foreign nucleic acid molecule codes for a
deregulated ADP-glucose pyrophosphorylase, particularly preferably
the ADP-glucose pyrophosphorylase from E. coli which is termed
glgC16 and which leads, on expression in transgenic potato plants,
to an increased starch synthesis rate. Cold-stored potato tubers of
these plants show a significantly reduced accumulation of hexoses
(Stark et al., Science 258, (1992), 287-292; Stark et al., Ann. NY
Acad. Sci. 792, (1996), 26-37).
[0155] In a further embodiment, the present invention relates to
the use of the above described plant material, which can be used in
the inventive process for producing heat-treated foods which have a
reduced acrylamide content compared with corresponding conventional
heat-treated foods.
[0156] In a further embodiment, the present invention relates to
the use of plant material which, compared with corresponding
conventional plant material, has a reduced content of soluble
sugars and/or amino acids for producing heat-treated foods having a
reduced acrylamide content.
[0157] In a further embodiment, the present invention relates to
the use of the above described plant material which can be used in
the inventive process for reducing the acrylamide content of
heat-treated foods.
[0158] In a further embodiment, the present invention relates to a
process for identifying plant material which is suitable for
producing heat-treated foods having a reduced acrylamide content,
which comprises: [0159] a) determining the content of soluble
sugars and/or amino acids in plant material which is suitable for
producing heat-treated foods; and [0160] b) selecting such plant
material according to process step a) which, compared with
corresponding conventional plant material, has a reduced content of
soluble sugars and/or amino acids.
[0161] All of the publications and patents referred to in the
specification are hereby incorporated by the reference in their
entirety.
Methods:
Determination of Glucose, Fructose and Sucrose:
[0162] To determine the contents of glucose, fructose and sucrose
in potato tubers, small pieces (diameter approximately 10 mm) of
potato tubers are frozen in liquid nitrogen and then extracted for
one hour at 80.degree. C. in 0.5 ml of 80% (vol./vol.) ethanol.
After centrifugation (3 min, 3 000 rpm), the supernatant is
withdrawn and the deposit is again extracted in 0.5 ml of 80%
(vol./vol.) ethanol. This process is repeated. The combined
supernatants are used to determine the amount of soluble
sugars.
[0163] Soluble glucose, fructose and sucrose are determined
quantitatively in an assay solution of the following
composition:
[0164] 100 mM imidazole/HCl (pH 6.9)
[0165] 5 mM MgCl.sub.2
[0166] 2 mM NAD.sup.+
[0167] 1 mM ATP
[0168] 200 .mu.l of sample
[0169] 2 units of glucose-6-phosphatedehydrogenase (from
Leuconostoc mesenteroides)
[0170] The assay solution is incubated at room temperature for 5
min. The sugars are then determined using customary photometric
methods by measuring the absorption at 340 nm after the successive
addition of
[0171] 1 500 units of yeast hexokinase (to determine glucose)
[0172] 2.5 units of yeast phosphoglucoisomerase (to determine
fructose)
[0173] 350 units of yeast p-fructosidase (to determine sucrose) to
a reaction volume of 200 .mu.l.
[0174] The examples below illustrate the invention:
EXAMPLE 1
Production of Potato Crisps and Potato Chips from Potato Tubers
[0175] To produce potato crisps and potato chips, ripe potato
tubers of transgenic potato plants which have a decreased
expression of the R1 gene (Lorberth et al., Nature Biotechnology
16, (1998), 473-477) and also potato tubers of potato plants which
have a decreased R1 gene expression and in addition a decreased
expression of branching enzyme I gene (WO97/11188) were used.
[0176] The crisps and chips were further processed immediately
after harvest and also after storage at 4.degree. C. for differing
times.
[0177] The tubers were peeled by hand and then sliced in a slicing
machine (model Chef200, from Saro Emmerich, Germany) into slices
for the production of crisps or cut using a punch (Weisser,
Germany) to form chips.
[0178] The samples were deep-fat fried in a deep-fat fryer (Frita4,
Franke, Frifri aro GmbH, Germany) for differing times using plant
fat (Palmaja, Meylip mbH & Co. KG, Germany) at a temperature of
180.degree. C.
[0179] The deep-fat fried products were then comminuted and
analyzed for their acrylamide content. This was detected using
GC/MS or LC/MS-MS after derivatization (Epa method 8032a, U.S.
Environmental Protection Agency). This ensures, in addition to a
low limited determination, a high selectivity of detection.
[0180] In the case of all deep-fat fried samples, it was found that
the acrylamide content in the transgenic potato tubers was reduced
by at least 15% compared with the acrylamide content of the tubers
of the corresponding wild type plants.
EXAMPLE 2
Determination of the Acrylamide Content of Potato Crisps and Chips
Produced From Potato Tubers Having Decreased R1- and Branching
Enzyme I-Gene Expression
[0181] The potato crisps and chips produced according to example 1
were analyzed for their acrylamide content.
[0182] Non-genetically-modified plants are termed hereinafter wild
type plants. The transgenic potato plants which have a decreased
expression of the R1 gene (Lorberth et al., Nature Biotechnology
16, (1998), 473477) and in addition a decreased branching enzyme I
gene expression (see international patent application WO 97/11188)
are termed hereinafter 015VL001.
[0183] If freshly harvested tubers of potato plants are used for
deep-fat frying at 180.degree. C. for 3 and 6 minutes, the potato
crisps have the following acrylamide content: TABLE-US-00001 TABLE
1 Percentage acrylamide content of crisps (produced from potato
tubers directly after harvesting). The wild type was set at 100%.
Crisps deep-fat frying time Crisps deep-fat frying time 3 min 6 min
Wild type 100% 100% 015VL001 31% 49%
[0184] The absolute acrylamide content increases greatly with
increasing deep-fat frying time. This is the case not only for
crisps from wild type tubers, but also for crisps from transgenic
tubers. For both deep-fat frying times, the increase in acrylamide
content in the crisps which were produced from the transgenic
potato tubers, however, is significantly reduced compared with the
crisps of wild type plants. At a deep-fat frying time of 3 min, the
acrylamide content in the transgenic crisps is reduced by
approximately 70% compared with the wild type crisps. At a deep-fat
frying time of 6 min, there is a reduction in acrylamide formation
in the transgenic crisps of approximately 50% compared with the
wild type.
[0185] In a further experiment, potato tubers stored at 4.degree.
C. were used for producing potato crisps. After harvest, the
transgenic tubers and the associated wild type tubers were stored
at 4.degree. C. for 56 days. Potato crisps and potato chips were
produced and deep-fat fried at 180.degree. C. for differing times
under the conditions described above: TABLE-US-00002 TABLE 2
Percentage acrylamide content of crisps (produced from tubers
stored at 4.degree. C.). The wild type was set at 100%. Crisps
deep-fat frying time Crisps deep-fat frying time 3 min 6 min Wild
type 100% 100% 015VL001 26% 28%
[0186] The absolute acrylamide content always greatly increases in
the products from potatoes stored at 4.degree. C. However, it is
shown that the acrylamide content in the crisps made from
transgenic potato tubers increases by approximately 70% less
compared with the crisps made from corresponding wild type plants,
both for a deep-fat frying time of 3 min, and for a deep-fat frying
time of 6 min.
[0187] In a further experiment, potato chips were produced from
cold-stored potato tubers (stored at 4.degree. C. for 56 days) as
described in example 1 and deep-fat fried. In contrast to the
potato crisps, the potato chips were pre-fried for 30 seconds at
180.degree. C., laid out on kitchen paper, and cooled to room
temperature and only then deep-fat fried for the specified time.
TABLE-US-00003 TABLE 3 Percentage acrylamide content of potato
chips (produced from cold-stored tubers). The wild type was set at
100%. Potato chips deep-fat frying Potato chips deep-fat frying
time 3 min time 6 min Wild type 100% 100% 015VL001 55% 42%
[0188] The absolute acrylamide contents are lower in the potato
chips compared with potato crisps. This is certainly primarily due
to the smaller surface area of the potato chips compared with
potato crisps per kg of potato. The percentage acrylamide contents,
in this product also, show a reduction in the potato chips made
from transgenic potato plants by approximately 50% at both deep-fat
frying times compared with potato chips made from wild type
tubers.
[0189] In the context of industrial production of crisps or potato
chips, the sliced potatoes were blanched before deep-fat frying.
The blanching can take place in a water or steam blancher. The
blanching conditions are not fixed values, but vary very greatly
depending on the quality of the potatoes used. During the
blanching, soluble sugars are partly washed out. This causes more
uniform browning of the potato products in deep-fat frying.
[0190] To demonstrate that the inventive process also leads to a
reduction in the acrylamide formation in the potato products made
from transgenic potato plants under changed process conditions,
compared with the products from corresponding wild type tubers, the
blanching was performed on a laboratory scale by washing the sliced
potatoes with hot mains water.
[0191] For this purpose, approximately 200 g of potatoes (stored at
4.degree. C. for 56 days after harvest) were washed three times,
each time using 5 litres of mains water at 45.degree. C., each time
for 1.5 minutes. The sliced potatoes were then dried on domestic
paper and deep-fat fried at 180.degree. C. for 3 minutes as
described above: TABLE-US-00004 TABLE 4 Percentage acrylamide
content of washed crisps (produced from potato tubers). The wild
type was set at 100%. Crisps deep-fat frying time 3 min Wild type
100% 015VL001 21%
[0192] The washing leads to a reduction in acrylamide formation in
crisps which were produced from potato tubers of wild type plants
by approximately 16% compared with unwashed potato crisps.
[0193] Compared with the "washed" crisps which were produced from
potato tubers of wild type plants, the "washed" crisps which were
produced from potato tubers of the transgenic potato plants have an
acrylamide formation which is decreased by approximately 80%.
[0194] In a further analysis, the contents of soluble sugars, in
particular glucose and/or fructose, were determined compared with
the corresponding conventional plant material of wild type
plants:
[0195] For this purpose the potato tubers were peeled and a sample
having a diameter of approximately 0.5 cm sample was cut out using
a cork borer (from Roth). From this sample a slice approximately 2
mm thick each time from the start, one quarter and one half from 5
different tubers in each case was combined in a reaction vessel and
used to determine soluble sugars.
[0196] The determination of the contents of sugars, in particular
fructose and glucose, of plant material is known to those skilled
in the art and was carried out as described above. TABLE-US-00005
TABLE 6 Comparison of the percentage soluble sugar contents of
fresh harvested and stored tuber samples based on tubers of the
corresponding wild type (100%). Wild type 015VL001 Wild type
015VL001 after after directly after directly after storage storage
harvest harvest at 4.degree. C. at 4.degree. C. Glucose 100% 61%
100% 48% [.mu.mol/g fresh weight] Fructose 100% 67% 100% 53%
[.mu.mol/g fresh weight] Sucrose 100% 110% 100% 69% [.mu.mol/g
fresh weight]
[0197] Storage causes a sharp rise in the contents of soluble
sugars in wild type plants. The tubers of line 015VL show, directly
after harvest, contents of glucose and fructose reduced by
approximately 30%-40% compared with wild type plants. After the
above described cold storage, there is reduction of glucose or
fructose of approximately 50% in the transgenic plants compared
with the corresponding wild type plants.
[0198] If the content of glucose or fructose is correlated with the
acrylamide content in crisps, it is seen that there is a linear
correlation between the content of glucose or fructose in the
potato tuber and the formation of acrylamide in the deep-fat fried
product crisps.
[0199] It is has thus been shown for the first time that there is a
correlation between the formation of acrylamide in heat-treated
foods and the content of soluble sugars of the plant material used
to produce the heat-treated food. The effect on reduction of
acrylamide formation is much more pronounced than expected.
EXAMPLE 3
Determination of the Acrylamide Content of Potato Crisps and Potato
Chips Produced From Potato Tubers Having Decreased R1-Gene
Expression
[0200] The potato crisps and potato chips produced according to
example 1, which were produced from potato tubers having decreased
R1 gene expression, were analyzed for their acrylamide content.
[0201] In this case, as already described in example 2, firstly
tests were made of differing deep-fat frying times, and also
samples which had been differently stored or washed were
studied.
[0202] The results described in example 2 were confirmed, that is
to say potato crisps and potato chips produced from potato tubers
having decreased R1 gene expression, likewise show, under the
conditions described in more detail in example 2, less acrylamide
compared with corresponding products which were produced from
potato tubers from corresponding non-genetically-modified wild type
plants.
EXAMPLE 4
Production of Differing Varieties of Transgenic Potato Plants
Having Reduced R1 Gene Expression
[0203] To produce transgenic potato plants having reduced R1 gene
expression, the T-DNA of plasmid IR5/29 was transferred to potato
plants of cultivars Tomensa, Solara and Bintje, using agrobacteria,
as described in Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29).
Notes on Vector IR5/29:
[0204] IR5-29 is a derivative of plasmid pGSV71 which contains,
inter alia, the sequence of the promoter of the patatin gene B33
from Solanum tuberosum (Rocha-Sosa et al., (1989), see above) and
the complete R1-cDNA (Lorberth et al. Nature Biotechnology 16,
(1998), 473-477) in the "sense" orientation to the promoter.
[0205] pGSV71 is a derivative of plasmid pGSV7, which is derived
from the intermediate vector pGSV1. pGSV1 is a derivative of
pGSC1700, the construction of which was described by Cornelissen
and Vanderwiele ((1989), Nuclear transcriptional activity of the
tobacco plastid psbA promotor. Nucleic Acids Research 17: 19-29).
pGSV1 was obtained from pGSC1700 by deletion of the carbenicillin
resistance gene and deletion of the T-DNA sequences of the TL-DNA
region of plasmid pTiB6S3.
[0206] pGSV7 contains the replication origin of plasmid pBR322
(Bolivar et al., (1977), Construction and characterization of new
cloning vehicles. II. A multipurpose cloning system. Gene, 2:
95-113) and the replication origin of the pseudomonas plasmid pVS1
(Itoh et al., (1984), Genetic and molecular characterization of the
Pseudomonas plasmid pVS1. Plasmid 11: 206-220).
[0207] pGSV7 also contains the selectable marker gene aadA from the
transposon Tn1331 from Klebsiella pneumoniae, which confers
resistance to the antibiotics spectinomycin and streptomycin
(Tolmasky, (1990), Sequencing and expression of aadA, bla, and tnpR
from the multiresistance transposon Tn1331. Plasmid. 24 (3):
218-226; Tolmasky and Crosa, (1993), Genetic organization of
antibiotic resistance genes (aac(6')-Ib, aadA, and oxa9) in the
multiresistance transposon Tn1331. Plasmid. 29 (1): 31-40).
[0208] The plasmid pGSV71 was obtained by cloning a chimeric bar
gene between the border regions of pGSV7. The chimeric bar gene
contains the promoter sequence of the cauliflower mosaic virus for
initiating transcription (Odell et al., (1985), Identification of
DNA sequences required for activity of the Cauliflower Mosaic Virus
35S promotor. Nature 313:
[0209] 810-812), the bar gene from Streptomyces hygroscopicus
(Thompson et al., (1987); Characterization of the herbicide
resistance gene bar from Streptomyces hygroscopicus. The EMBO
Journal, 6: 2519-2523) and the untranslated 3' region of the
nopaline synthase gene of T-DNA of pTiT37 for termination of
transcription and polyadenylation. The bar gene confers tolerance
towards the herbicide glufosinate ammonium.
[0210] The T-DNA contains the following elements in the order
cited: [0211] the left border sequence of the TL-DNA from pTiB6S3
(Gielen et al., (1984), The complete nucleotide sequence of the
TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. The EMBO
J. 3:835-846). [0212] the promoter of the patatin gene B33 from
Solanum tuberosum (Rocha-Sosa et al., 1989, see above) in a sense
orientation based on the left border sequence of the TL-DNA [0213]
the complete R1-cDNA (Lorberth et al., (1998), see above) in a
sense orientation based on the patanin promoter [0214] the
polyadenylation signal (3' end) of the octopine synthase gene (gene
3) of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984),
see above) in a sense orientation based on the left border sequence
of the TL-DNA [0215] the TaqI fragment of the non-translated 3' end
of the nopaline synthase gene (3' nos) from the T-DNA of plasmid
pTiT37 (Depicker et al., (1982), Nopaline synthase: transcript
mapping and DNA sequence. Journal of molecular and applied Genetics
1: 561-573) in an antisense orientation based on the left border
sequence of the TL-DNA [0216] the coding sequence of the
phosphinothricin resistance gene (bar) from Streptomyces
hygroscopicus (Thompson et al. (1987, see above) in an antisense
orientation based on the left border sequence of the TL-DNA. The
two terminal codons at the 5' end of the bar wild type gene were
replaced by the codons ATG and GAC. [0217] the P35S3 promoter
region of cauliflower mosaic virus (Odell et al., (1985), see
above) in an antisense orientation based on the left border
sequence of the TL-DNA [0218] the right border sequence of the
TL-DNA from plasmid pTiB6S3 (Gielen et al., (1984), see above.
[0219] After the various potato cultivars had been transformed,
Western blot analysis (Lorberth et al., Nature Biotechnology 16,
(1998), 473-477) was used to identify, for each of the cultivars,
various lines whose tubers had a markedly reduced amount of R1
protein, owing to a cosuppression effect.
[0220] The plants of cultivar Tomensa obtained by transformation
using plasmid IR5/29 were termed 093IR plants, those of cultivar
Solara were termed 095IR plants, and those of cultivar Bintje were
termed 092IR plants.
[0221] Potato tubers from lines 093IR360, 095IR049 and 092IR002
were used to produce potato chips (example 5).
EXAMPLE 5
Determination of the Acrylamide Content of Potato Chips Which Had
Been Produced From Different Varieties of Potato Tubers Having
Decreased R1 Gene Expression
[0222] Freshly harvested potato tubers of the plants produced
according to example 4 were processed to potato chips in accordance
with example 1 and pre-deep-fat fried according to example 2 for 30
seconds at 180.degree. C., laid out on kitchen paper and cooled to
room temperature and then deep-fat fried at 180.degree. C. for 3
minutes.
[0223] The potato chips produced had the following acrylamide
contents: TABLE-US-00006 TABLE 1 Percentage acrylamide content of
potato chips (produced from potato tubers directly after harvest).
Each corresponding wild type was set at 100%. Wild type Wild type
Wild type Tomensa 093IR360 Solara 095IR049 Bintje 092IR002
Acrylamide 100% 62% 100% 56% 100% 64% content [%]
[0224] The absolute acrylamide contents of the potato chips
produced in part vary considerably between the cultivars used. This
is primarily due to the differing absolute values of soluble
sugars. For instance potato chips from cultivar Solara, for
example, exhibited not only the highest acrylamide contents but
also the highest soluble sugar contents.
[0225] The relative acrylamide contents of the potato chips,
however, showed for all transgenic cultivars used a considerable
reduction in the amount of acrylamide by approximately 40-50%
compared with potato chips which had been produced from wild type
tubers.
[0226] In a further analysis, the contents of soluble sugars, in
particular glucose, fructose and sucrose, were determined in the
potato tubers from the various cultivars:
[0227] For this purpose the potato tubers were peeled in accordance
with example 2 and using a cork borer (from Roth), a sample of
diameter approximately 0.5 mm was cut out. From this cork borer
sample, an approximately 2 mm thick slice was taken in each case
from the start, one quarter and half way, from each of 5 different
tubers and combined in a reaction vessel and used for determining
soluble sugars.
[0228] The contents of sugars, in particular fructose and glucose,
of plant material were determined as described above.
[0229] As mentioned above, the absolute values of soluble sugars
vary greatly between the cultivars studied.
[0230] Cultivar Solara shows the highest glucose, fructose and
sucrose contents. Tomensa shows the lowest glucose and fructose
contents, and Bintje the lowest sucrose contents. Tubers from line
093IR360, directly after harvest, show glucose and sucrose contents
reduced by approximately 30-40% compared with the corresponding
wild type plants. Tubers of line 095IR049, directly after harvest,
show glucose and fructose contents reduced by approximately 10-30%
compared with wild type plants.
[0231] If the total glucose and/or fructose content is correlated
with the acrylamide content in potato chips, it may be seen that
there is a linear correlation between the glucose and/or fructose
content and the formation of acrylamide in the potato chips.
[0232] It was thus confirmed for various cultivars that the use of
potato plants having decreased R1 gene expression permits the
production of heat-treated foods, in particular potato chips, which
are distinguished by a markedly reduced acrylamide content compared
with corresponding heat-treated foods which are produced from
corresponding non-genetically-modified wild type plants.
EXAMPLE 6
Determination of the Acrylamide Content of Potato Chips Produced
From Stored Potato Tubers of Differing Varieties of Reduced R1-Gene
Expression
[0233] Potato tubers stored at 4.degree. C. for 73 days from the
plants produced in accordance with Example 4 were processed to
potato chips in accordance with Example 1 and deep-fat prefried at
180.degree. C. for 30 seconds in accordance with Example 2, placed
on kitchen paper and cooled to room temperature, and then deep-fat
fried at 180.degree. C. for 3 minutes.
[0234] The potato chips produced had the following acrylamide
contents: TABLE-US-00007 TABLE 1 Percentage acrylamide content in
potato chips (produced from potato tubers stored at 4.degree. C.).
The corresponding respective wild types were set at 100%. Wild type
Wild type Wild type Tomensa 093IR360 Solara 095IR049 Bintje
092IR002 Acrylamide 100% 55% 100% 70% 100% 58% content [%]
[0235] The absolute acrylamide content in the products from
potatoes stored at 4.degree. C. always increases markedly. However,
it is found for the varieties listed here, also, that the
acrylamide content in the potato chips from transgenic potato
tubers increases by approximately 30-45% less compared with the
potato chips from corresponding wild type plants.
[0236] In a further experiment, potato tubers of the cultivar
Desiree having reduced R1-gene expression (Lorberth et al., Nature
Biotechnology 16, (1998), 473-477) were stored at 4.degree. C. for
73 days. Potato chips and crisps were produced as described in
Example 1. Crisps were deep-fat fried for 3 minutes at 180.degree.
C. as described in Example 2. In contrast to the potato crisps,.
potato chips were deep-fat pre-fried at 180.degree. C. for 30
seconds, placed on kitchen paper and cooled to room temperature and
then deep-fat fried for 3 minutes.
[0237] The potato chips and crisps produced had the following
acrylamide contents: TABLE-US-00008 TABLE 2 Percentage acrylamide
content of potato chips and crisps (produced from tubers stored at
4.degree. C.). The wild type was set at 100%. Potato chips Crisps
deep-fat frying time deep-fat frying time 3 min 3 min Wild type
100% 100% 009VL045 56% 33%
[0238] The absolute acrylamide content in the products from
potatoes stored at 4.degree. C. always increases markedly. However,
it is also found in this experiment that the acrylamide content
increases by approximately 70% less in the crisps made from
transgenic potato tubers compared with the crisps made from the
corresponding wild type plants.
[0239] In a further experiment, potato tubers of cultivar Desiree
of reduced R1-gene expression (Lorberth et al., Nature
Biotechnology 16, (1998), 473-477) were stored at 8.degree. C. for
73 days. These stored potato tubers were processed into potato
chips in accordance with Example 1 and deep-fat pre-fried for 30
seconds at 180.degree. C. in accordance with Example 2, placed on
kitchen paper and cooled to room temperature and then deep-fat
fried at 180.degree. C. for 3 minutes.
[0240] The potato chips produced had the following acrylamide
contents: TABLE-US-00009 TABLE 3 Percentage acrylamide content of
potato chips (produced from tubers stored at 8.degree. C.). The
wild type was set at 100%. Potato chips deep-fat frying time 3 min
Wild type 100% 009VL045 52%
[0241] The absolute acrylamide content in the products from potato
tuber stored at 8.degree. C. does not increase as greatly as in the
case of potato tubers stored at 4.degree. C. However, in this
experiment also, it is found that the acrylamide content in the
potato chips from transgenic potato tubers increases by
approximately 48% less compared with potato chips made from
corresponding wild type plants.
* * * * *
References